NSCR

NSCR

Nascent Strand Capture and Release (NSCR) isolates and sequences origin of DNA replication by capturing short nascent strands (SNS) which are single-stranded RNA-DNA chimeras . In short, NSCR captures and purifies RNA-DNA chimeric SNS, and releases the DNA part by cutting the strand at the end of the initial RNA primer section.

First, genomic DNA are denatured, size selected through a sucrose gradient for 400 to 2000 nt fragments, and 5' biotinylated. Biotinylated fragments are then isolated by streptavidin pull-down, purifying both SNS and gDNA strands. Here, RNase I is used to separate the DNA from the initial RNA primer section of the single-stranded RNA-DNA strand. This leaves RNA primers and gDNA on the streptavidin beads. Released nascent DNA strands are amplified and sequenced. This method is an improvement over widely-use SNS purification methods using BrdU and lambda-exonuclease.

Pros:

• Maps genome-wide DNA replication origins
• Higher discovery of nascent strands than lexo-based methods (2231 vs. 3922 peaks)
• RNase I cleaves with minimal sequence specificity
• Can be used for systems that use small RNA primers to produce RNA-DNA chimeras

Cons:

• Less than 10% purification yield (1-2 ng of SNS from 1 mg total DNA)
• False reads may arise due to incorporation of rNTPs by ribonucleotides into the SNSs

• NSCR: Kunnev D., Freeland A., Qin M., Wang J. and Pruitt S. C. (2015) Isolation and sequencing of active origins of DNA replication by nascent strand capture and release (NSCR). J Biol Methods 2:

1. Kunnev D., Freeland A., Qin M., Wang J. and Pruitt S. C. (2015) Isolation and sequencing of active origins of DNA replication by nascent strand capture and release (NSCR). J Biol Methods 2: